Forward genetic screening using fundus spot scale identifies an essential role for Lipe in murine retinal homeostasis.

Microglia play a role in the pathogenesis of many retinal diseases. Fundus spots in mice often correlate with the accumulation of activated subretinal microglia. Here we use a semiquantitative fundus spot scoring scale in combination with an unbiased, state-of-the-science forward genetics pipeline to identify causative associations between chemically induced mutations and fundus spot phenotypes. Among several associations, we focus on a missense mutation in Lipe linked to an increase in yellow fundus spots in C57BL/6J mice. Lipe-/- mice generated using CRISPR-Cas9 technology are found to develop accumulation of subretinal microglia, a retinal degeneration with decreased visual function, and an abnormal retinal lipid profile. We establish an indispensable role of Lipe in retinal/RPE lipid homeostasis and retinal health. Further studies using this new model will be aimed at determining how lipid dysregulation results in the activation of subretinal microglia and whether these microglia also play a role in the subsequent retinal degeneration.

Forward genetic analysis using OCT screening identifies Sfxn3 mutations leading to progressive outer retinal degeneration in mice.

Retinal disease and loss of vision can result from any disruption of the complex pathways controlling retinal development and homeostasis. Forward genetics provides an excellent tool to find, in an unbiased manner, genes that are essential to these processes. Using N-ethyl-N-nitrosourea mutagenesis in mice in combination with a screening protocol using optical coherence tomography (OCT) and automated meiotic mapping, we identified 11 mutations presumably causative of retinal phenotypes in genes previously known to be essential for retinal integrity. In addition, we found multiple statistically significant gene-phenotype associations that have not been reported previously and decided to target one of these genes, Sfxn3 (encoding sideroflexin-3), using CRISPR/Cas9 technology. We demonstrate, using OCT, light microscopy, and electroretinography, that two Sfxn3-/- mouse lines developed progressive and severe outer retinal degeneration. Electron microscopy showed thinning of the retinal pigment epithelium and disruption of the external limiting membrane. Using single-cell RNA sequencing of retinal cells isolated from C57BL/6J mice, we demonstrate that Sfxn3 is expressed in several bipolar cell subtypes, retinal ganglion cells, and some amacrine cell subtypes but not significantly in Müller cells or photoreceptors. In situ hybridization confirmed these findings. Furthermore, pathway analysis suggests that Sfxn3 may be associated with synaptic homeostasis. Importantly, electron microscopy analysis showed disruption of synapses and synaptic ribbons in the outer plexiform layer of Sfxn3-/- mice. Our work describes a previously unknown requirement for Sfxn3 in retinal function.

Mice With a Combined Deficiency of Superoxide Dismutase 1 (Sod1), DJ-1 (Park7), and Parkin (Prkn) Develop Spontaneous Retinal Degeneration With Aging.

Purpose: Chronic oxidative stress is an important mechanism of disease in aging disorders. We do not have a good model to recapitulate AMD and other retinal disorders in which chronic oxidative stress plays an important role. We hypothesized that mice with a combined deficiency in superoxide dismutase 1 (Sod1), DJ-1 (Park-7), and Parkin (Prkn) (triple knock out, TKO) would have an increased level of chronic oxidative stress in the retina, with anatomic and functional consequences just with aging. Methods: Eyes of TKO and B6J control mice were (1) monitored with optical coherence tomography (OCT) and electroretinography (ERG) over time, and (2) collected for oxidative marker protein analysis by ELISA or immunohistochemistry and for transmission electron microscopy studies. Results: TKO mice developed qualitative disruptions in outer retinal layers in OCT by 3 months, increased accumulation of fundus spots and subretinal microglia by 6 months of age, significant retinal thinning by 9 months, and decreased ERG signal by 12 months. Furthermore, we found increased accumulation of the oxidative marker malondialdehyde (MDA) in the retina and increased basal laminal deposits (BLD) and mitochondria number and size in the retinal pigment epithelium of aging TKO mice. Conclusions: TKO mice can serve as a platform to study retinal diseases that involve chronic oxidative stress, including macular degeneration, retinal detachment, and ischemic retinopathies. In order to model each of these diseases, additional disease-specific catalysts or triggers could be superimposed onto the TKO mice. Such studies could provide better insight into disease mechanisms and perhaps lead to new therapeutic approaches.

Relationship between ADAMTS4 and carotid atherosclerotic plaque vulnerability in humans.

BACKGROUND: Rupture of atherosclerotic plaques and the resulting thrombosis are vital causes of clinical ischemic events. Recent studies have shown that ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) is a pathogenic factor of plaque vulnerability in mice. However, the relationship between ADAMTS4 and carotid atherosclerotic vulnerable plaques in humans remains unclear. METHODS: Forty-eight carotid atherosclerotic plaque specimens were obtained from 48 carotid artery stenosis inpatients undergoing carotid endarterectomy. We performed hematoxylin and eosin and Movat pentachrome staining for histologic characteristics; immunohistochemical staining for ADAMTS4, versican, and macrophages; and serologic tests for ADAMTS4. Patients were divided into stable and vulnerable groups on the basis of histologic characterization according to the classification criteria of the American Heart Association. Comparison between the groups was carried out using SPSS 17.0 (SPSS Inc, Chicago, Ill). RESULTS: Expression of ADAMTS4 in the plaque and its serum concentration were significantly higher in the vulnerable group compared with the stable one (P = .004 and P = .021, respectively), whereas the expression of versican was lower in the vulnerable group than in the stable group (P = .015). Univariate analysis revealed that the incidence of symptomatic cerebral ischemic events and ADAMTS4 serum levels were statistically higher in the vulnerable group compared with the stable group (P = .021 and P = .029, respectively). Multivariate analysis showed that ADAMTS4 was an independent risk factor (odds ratio, 1.14; P = .038). CONCLUSIONS: Our study revealed that ADAMTS4 expression was upregulated during carotid atherosclerotic plaque development. Serum levels of ADAMTS4 were associated with increased plaque vulnerability in both symptomatic and asymptomatic patients with carotid artery stenosis. ADAMTS4 may be a potential biomarker for plaque vulnerability.

Increased susceptibility to fundus camera-delivered light-induced retinal degeneration in mice deficient in oxidative stress response proteins.

Oxidative stress is an important contributor to the pathogenesis of many retinal diseases including age-related macular degeneration and retinal dystrophies. Light-induced retinal degeneration (LIRD) can serve as a model in which to study the response of the retina to stress. Of note, many genetic mutant mice are in a C57BL/6 J background and are thus resistant to the usual LIRD models. We recently developed a new model of fundus camera-delivered light-induced retinal degeneration (FCD-LIRD) which is effective in strains of mice expressing the light-resistant variant of RPE65 (450Met), including C57BL/6 J. In this work we investigated whether FCD-LIRD would be useful as a model in which to test the effect of genetic mutations on the response of the retina to stress. Furthermore, we tested whether oxidative stress plays an important role in the setting of this new FCD-LIRD model. FCD-LIRD was applied to C57BL/6 J mice and to mice simultaneously deficient in three proteins that are important in the response of the retina to oxidative stress (SOD1, DJ-1 and Parkin). Using fundus photography, we found that retinal damage was dramatically increased in the SOD1/DJ-1/Parkin deficient mice compared to C57BL/6 J. Outer retinal OCT volume and RPE cell morphology analysis in ZO-1-stained flat mounts added support to these findings. Gene expression analysis confirmed a strong oxidative stress response after FCD-LIRD, which was differentially altered in the SOD1/DJ1/Parkin deficient mice. We conclude that FCD-LIRD is useful to study the effect of genetic mutations on the response of the retina to light stress in light-resistant strains of mice. Furthermore, oxidative stress seems to be an important component of FCD-LIRD. Finally, we have established protocols to quantify the effect of FCD-LIRD on the retina and RPE which will be useful for future studies. Further dissection of the mechanisms by which the retina responds to light-induced oxidative stress may result in new strategies to modulate this response, which could lead to a reduction in retinal and RPE damage.

Fundus Camera-Delivered Light-Induced Retinal Degeneration in Mice With the RPE65 Leu450Met Variant is Associated With Oxidative Stress and Apoptosis.

PURPOSE: Oxidative stress, partly due to light, has an important role in many retinal diseases, including macular degeneration and retinal dystrophies. The Leu450Met variant of RPE65 is expressed in C57BL/6 and in many genetically modified mice. It confers significant resistance to light induced retinal degeneration (LIRD). Our goal was to develop an effective and efficient method to induce LIRD in resistant mice that would recapitulate mechanisms seen in known models of LIRD. METHODS: The retinas of C57BL/6J mice were exposed to light using a murine fundus camera. Two protocols (with and without intraperitoneal fluorescein) were used. Optical coherence tomography (OCT) helped determine the location and extent of retinal damage. Histology, TUNEL assay, quantitative (q) PCR, and immunohistochemistry were performed. RESULTS: Both protocols consistently generated LIRD in C57BL/6J mice. Optical coherence tomography and histology demonstrated that retinal damage starts at the level of the photoreceptor/outer retina and is more prominent in the superior retina. Fundus camera-delivered light-induced retinal degeneration (FCD-LIRD) is associated with apoptosis, subretinal microglia/macrophages, increased expression of oxidative stress response genes, and C3d deposition. CONCLUSIONS: We characterize two new models of light-induced retinal degeneration that are effective in C57BL/6J mice, and can be modulated in terms of severity. We expect FCD-LIRD to be useful in exploring mechanisms of LIRD in resistant mice, which will be important in increasing our understanding of the retinal response to light damage and oxidative stress.

Evaluation of urine fibrinogen level in a murine model of contrast-induced nephropathy.

OBJECTIVE: The mechanisms of contrast-induced nephropathy are not fully understood and sensitive biomarkers of contrast-induced nephropathy are yet to be found. We investigated whether urinary fibrinogen could be a potential biomarker for contrast-induced nephropathy. METHODS: To create a contrast-induced nephropathy model, mice received a prostaglandin synthesis inhibitor (indomethacin) and a nitric oxide synthase inhibitor (Nω-Nitro-L-arginine methyl ester) intraperitoneally followed by a different dose of iodixanol. In the control group, normal saline was administered. Urinary fibrinogen and serum creatinine were analyzed using enzyme-linked immunosorbent assay. Kidneys were used to quantify fibrinogen using qRT-PCR and Western blot and for histopathological examination. RESULTS: Histopathological examination demonstrated mild renal injury in the low-dose group, and moderate renal injury in the high-dose group. Urinary fibrinogen levels were significantly increased in an iodixanol dose-dependent manner (control vs. low-dose group, P < 0.05; control vs. high-dose group P < 0.01). Serum creatinine levels were only increased in the high-dose group (P < 0.01 compared to control), but not in the low-dose group. For fibrinogen-gene expression, in the low-dose group, Fgγ increased (qRT-PCR, Western blot, P < 0.05) in the high-dose group, Fgß and Fgγ decreased (qRT-PCR, P < 0.01; Western blot, P < 0.05), and Fgα increased (qRT-PCR, P < 0.05; Western blot, P < 0.05). CONCLUSIONS: We propose that urinary fibrinogen could be used as a potential biomarker for early contrast-induced nephropathy diagnosis.

Grading system modification and management of blunt aortic injury.

BACKGROUND: The traditional approach to blunt aortic injury (BAI) has been emergent intervention. This study aimed to utilize a modified imaging grading system that may allow us to categorize these injuries as needing emergent, urgent, or non-operative management. METHODS: From January 2003 to December 2011, 28 patients with BAI were managed at our institution. Imaging and medical records were reviewed retrospectively. BAI was classified into 4 grades based on imaging studies. Grade Ia: intimal tear, Grade Ib: intramural hematoma; Grade II: intimal injury with periaotic hematoma; Grade IIIa: aortic transection with pseudoaneurysm, Grade IIIb: multiple aortic injuries; and Grade IV: free rupture. Progression and clinical outcomes of ABI were analyzed. RESULTS: Of the 28 patients, 22 were males and 6 were females with mean age of 38 (range, 7 - 69) years. Twenty-five (89.3%) had descending thoracic aortic injury, two (7.1%) had abdominal aortic injury and one (3.6%) presented with multiple aortic injuries. Three patients (10.7%) with Grade I, 1 (3.6%) Grade II, 22 (78.6%) Grade III, and 2 (7.1%) Grade IV injuries. Twenty-five patients underwent thoracic endovascular aortic repair and 3 were managed medically. Median time between injury and surgical intervention was (2 ± 1) days. One (3.6%) patient developed paraplegia after thoracic endovascular aortic repair (TEVAR). One Type 2 endoleak spontaneously sealed within 1 month, and another patient died from ruptured Type 1 endoleak 3 years later. Median follow-up time was 16 (range, 1 - 96) months. Perioperative 30-day mortality rate was 3.6%. CONCLUSIONS: This study based on our modified BAI grading system indicated that Grade I BAI can be managed conservatively. Grade II injury requires close observation and repeated computerized tomography angiogram (CTA) within 48 - 72 hours. If injury appears worse on follow up imaging, surgery should be performed. Delayed repair of Grade III BAI is acceptable if associated life threatening traumatic injuries need to be addressed first.

Experience with a new negative pressure incision management system in prevention of groin wound infection in vascular surgery patients.

OBJECTIVE: Groin wound infection is an important cause of postoperative morbidity in vascular surgery patients, especially when prosthetic grafts are involved. The objective of this study was to investigate if Prevena (Kinetic Concepts, Inc, San Antonio, Tex), a negative pressure incision management system, could reduce the risk of groin wound infection in patients after vascular surgery. METHODS: Ninety patients (115 groin incisions) underwent longitudinal or transverse femoral cutdown for vascular procedures. A retrospective chart review was performed on 63 consecutive incisions in patients in the non-Prevena group from December 2009 to November 2010 and on 52 consecutive incisions in patients in the Prevena group from January 2011 to December 2011. Prevena was applied intraoperatively and removed 5 to 7 days postoperatively. The non-Prevena group received either a skin adhesive or absorbent dressing. Groin incisions were assessed, and infection was graded based on Szilagyi classifications. Student t-test and two-sample proportion z test were used for statistical analyses. A P value < .05 was considered statistically significant. RESULTS: Comorbidities and known risk factors for infection were compared; there were no statistically significant differences between the two groups. Prosthetic material was used in 34 (65%) incisions in the Prevena group and 29 (46%) incisions in the non-Prevena group. Fifty (96%) incisions within the Prevena group and 60 (96%) in the non-Prevena group were classified as clean surgical wounds. Wounds were evaluated at 7 days and 30 days postoperatively. Of 63 groin incisions in 49 patients in the non-Prevena group, 19 (30%) incisions had groin wound infections. Wound infections were classified into Szilagyi grade I (10; 16%), Szilagyi grade II (7; 11%), and Szilagyi grade III (2; 3%). Of 52 groin incisions in 41 patients in the Prevena group, three (6%) incisions had Szilagyi grade I wound infections. No grade II or III infections occurred in this group. Overall incidence of infection between the two groups was statistically significant (P = .0011). CONCLUSIONS: In this clinical study, Prevena negative pressure dressing significantly decreased the incidence of groin wound infection in patients after vascular surgery.

Accuracy of digital subtraction angiography, computed tomography angiography, and magnetic resonance angiography in grading of carotid artery stenosis in comparison with actual measurement in an in vitro model.

BACKGROUND: The aim of this study was to investigate the accuracy of digital subtraction angiography (DSA), computed tomography angiography (CTA), and magnetic resonance angiography (MRA) in grading of carotid stenosis compared with actual measurement in an in vitro model. METHODS: Various grades of stenosis were created by adhering different amounts of silicone rubber sealant onto the inner wall of clear, radiolucent tubes. After DSA, CTA, and MRA, the tubes were transected with 1-mm interval through the plaques. The cross-sectional areas were digitally photographed, and the percentage of area reduction of every single slide was measured with ImageJ planimetric software. The maximum actual area reduction (AAR) stenosis of each tube was recorded. The differences among DSA, CTA, MRA, and AAR were compared statistically using paired Student t test. RESULTS: Overall, CTA and MRA significantly underestimated the degrees of stenosis compared with AAR (P = 0.001 and P = 0.0009, respectively), and no significant difference was found between DSA and AAR (P = 0.40). In the subgroup with stenosis of <70%, there was no significant difference between DSA, CTA, and MRA versus AAR (P = 0.18, P = 0.16, and P = 0.08, respectively). In the subgroup with severe stenosis of >70%, CTA and MRA significantly underestimated the stenosis versus AAR (P = 0.004, and P = 0.007 respectively), and DSA significantly overestimated the stenosis (P = 0.0007). CONCLUSIONS: This in vitro model study demonstrated that CTA and MRA underestimate the lesions in severe stenosis of >70%. DSA tends to overestimate the disease. The accuracy of DSA is affected by plaque morphology, such as mountain-shaped lesions.